Thermostable alpha amylase
a technology of alpha-amylase and amylase, applied in hydrolases, biochemistry apparatus and processes, enzymes, etc., can solve the problems of increased alpha-amylase and protease activity, pre-harvest sprouting of grain, poor baking performance, etc., and achieve the effect of reducing the number of stirrings or falling the number of flour
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example 1 preparation
of a Polypeptide According to the Invention
[0210]A polypeptide according to the invention, further referred to as AMY 1, is comprised in SEQ ID No. 1. This sequence consists of a signal sequence of 17 amino acids for efficient secretion and a deduced mature polypeptide sequence of 478 amino acids (position 18-495 from SEQ ID No. 1). The signal sequence used for secretion of the AMY1 polypeptide is the pectin methyl esterase (pmeA) signal sequence (in position 1-17 in SEQ ID NO.1). The secreted (mature) AMY1 polypeptide of the invention is shown as position 18-495 of SEQ ID No. 1. To be able to express the polypeptide, a codon-adapted DNA sequence for expression of the polypeptide protein in Aspergillus niger was designed containing additional restriction sites for subcloning in an Aspergillus expression vector. Codon adaptation was performed as described in WO 2008 / 000632. The codon optimized DNA sequence for A. niger of the gene encoding the polypeptide of SEQ ID. NO. 1 is shown in...
example 2
Thermostability Testing of AMY1 Polypeptide According to the Invention. (Residual Activity)
[0216]Thermostability analyses of AMY1 polypeptide was performed by measuring Residual alpha amylase activity of the samples as described in Materials & Methods. A. niger alpha amylase (as set out in amino acids 18 to 495 of FIG. 3, as set out in amino acids 18 to 495 of SEQ ID No. 3) was used as a reference. All samples were incubated at elevated temperatures in the range of 51 to 70 degrees C. at pH 5.2. The results are depicted in Table 1 and FIG. 5 and show that AMY1 is significantly more thermostable than A. niger alpha amylase (ANA). After 15 min at 54.6 degrees C. AMY1 still has at least 100% of its alpha amylase activity, compared to less than 50% for the A. niger alpha amylase (ANA).
TABLE 1Residual alpha amylase activity at elevated temperatureT (° C.)(15 min, pH 5.2)ANAAMY15179%104%52.966%106%54.647%104%56.624%101%59.55%96%61.71%92%63.71%86%661%n.d.68n.d.48%70n.d.22%71.1n.d.8%72.1n.d...
example 3
Alpha Amylase Activity at Different Temperatures (Relative Activity)
[0217]Alpha amylase activity at different temperatures was measured as described above and the Relative Activity was determined, whereby the activity at 37 degrees C. was set at 100%. Results are presented in Table 2 and show that AMY1 has lower relative alpha amylase activity than A. niger alpha amylase (ANA) at 25 and 30 degrees C. The Act25 / Act37 ratio for AMY1 is 40% (versus 56% for A. niger alpha amylase) and the Act30 / Act37 ratio is 60% for AMY1 (versus 77% for A. niger alpha amylase).
TABLE 2Alpha amylase activity at different temperatures(Relative Activity, with the activity at 37 degrees C. set at 100%).ratioANAAMY1Act25 / Act37 ratio56%40%Act30 / Act37 ratio77%60%
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